PROJECT SUMMARY Successful completion of spermatogenesis relies upon precise spatiotemporal expression of distinct subsets of differentiation markers within the seminiferous epithelium. Failure to express genes at the correct time leads to arrested spermatogenesis and male infertility. The transcriptional mechanisms regulating this process, however, are not well understood. Our work has established that RNA Pol II pausing is critical for maintaining precise spatiotemporal gene expression during spermatogenesis. Paused RNA Pol II at the promoter ensures precise and rapid onset of gene transcription. This mechanism is particularly relevant to spermatogenesis wherein synchronous transcription of cohorts of genes is critical for morphogenesis and differentiation. The central hypothesis of this proposal is that RNA Pol II pausing occurs genome-wide in germ cells and is critical for the success of spermatogenesis. We have identified the TAR DNA binding protein of 43 kD (TDP-43) as a key player in maintaining paused Pol II at a target gene promoter in male germ cells. A corollary is that TDP-43 regulates pausing of a subset of genes in germ cells. TDP-43 is evolutionarily conserved and expressed in the mouse and human testis. We have found that TDP-43 is essential for spermatogenesis; conditional knockout of TDP-43 in germ cells led to maturation arrest and male infertility. Specific Aim 1 will test the overarching hypothesis that Pol II pausing is a key regulator of spatiotemporal gene transcription by determining the genome-wide occupancy of Pol II pause machinery in germ cells and seek biochemical validation for pausing. Aim 2 will study the mechanism of Pol II pausing in germ cells; test the hypothesis that TDP-43 recruits the pause machinery to a subset of promoters, map its interactions with pause factors and determine functional significance. Specific Aim 3 will test the hypothesis that loss of TDP-43 disrupts Pol II pausing at TDP-43 target promoters in germ cells leading to male infertility. In human cells including embryonic stem cells, Pol II pausing has been shown to play a pivotal role in regulation of gene transcription. The present study is significant because for the first time it will expand knowledge on a mechanism regulating the male germ cell transcriptome. The outcome will have a major impact on the understanding of the genetic basis of idiopathic male infertility. Thus, the proposed studies are aligned with high priority topic areas identified by the Fertility and Infertility (FI) Branch of NICHD: Genetic basis of idiopathic infertility and Models for infertility.